Extracting immunological and clinical heterogeneity across autoimmune rheumatic diseases by cohort-wide immunophenotyping

Objective Extracting immunological and clinical heterogeneity across autoimmune rheumatic diseases (AIRDs) is essential towards personalised medicine. Methods We conducted large-scale and cohort-wide immunophenotyping of 46 peripheral immune cells using Human Immunology Protocol of comprehensive 8-colour flow cytometric analysis. Dataset consisted of >1000 Japanese patients of 11 AIRDs with deep clinical information registered at the FLOW study, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). In-depth clinical and immunological characterisation was conducted for the identified RA patient clusters, including associations of inborn human genetics represented by Polygenic Risk Score (PRS). Results Multimodal clustering of immunophenotypes deciphered underlying disease-cell type network in immune cell, disease and patient cluster resolutions. This provided immune cell type specificity shared or distinct across AIRDs, such as close immunological network between mixed connective tissue disease and SLE. Individual patient-level clustering dissected patients with AIRD into several clusters with different immunological features. Of these, RA-like or SLE-like clusters were exclusively dominant, showing immunological differentiation between RA and SLE across AIRDs. In-depth clinical analysis of RA revealed that such patient clusters differentially defined clinical heterogeneity in disease activity and treatment responses, such as treatment resistance in patients with RA with SLE-like immunophenotypes. PRS based on RA case–control and within-case stratified genome-wide association studies were associated with clinical and immunological characteristics. This pointed immune cell type implicated in disease biology such as dendritic cells for RA-interstitial lung disease. Conclusion Cohort-wide and cross-disease immunophenotyping elucidate clinically heterogeneous patient subtypes existing within single disease in immune cell type-specific manner.


Supplementary Method of the immuno-phenotyping.
Informed consent having been obtained, peripheral blood samples were collected and promptly subjected to multicolor flow cytometry analysis.Peripheral blood mononuclear cells (PBMCs) were isolated from these samples using lymphocyte isolation medium (ICN/Cappel Pharmaceuticals, Aurora, OH, USA).Subsequently, the PBMCs were resuspended in phosphate-buffered saline (PBS) containing 3% human IgG (Baxter International Inc.) to inhibit Fc receptor activity and prevent nonspecific antibody binding.They were then incubated for 15 minutes at 48°C in a light-free environment, followed by a PBS wash containing 1% BSA.
The assessment of background fluorescence was accomplished through suitable isotypeand fluorochrome-matched control monoclonal antibodies.After antibody staining, the PBMCs were subjected to multicolor flow cytometry analysis using the FACSVerse (BD Biosciences, San Jose, CA, USA).The antibodies used for antibody staining are detailed in Supplementary Table 1.
For immune cell subset phenotyping, the comprehensive eight-color flow cytometry analysis protocol proposed by the National Institutes of Health/Federation of Clinical Immunology Societies (HIP protocol) was followed, with minor modification made to enable the detection of Tfh cells.Supplementary Figure 1 provides  To normalize potential batch effects among the samples and assays, we developed a pipeline to apply automated counting of the immune cells using R Bioconductor packages of openCyto (version 2.2.0).The list of the 46 cell types is shown in Supplementary Table 2.
a detailed representation of the gating strategy, which outputs the 46 immune cell types of the participants.The phenotypes of immune cell subsets were defined based on the Human Immunology Protocol of comprehensive 8-color flow cytometric analysis proposed by National Institutes of Health/Federation of Clinical Immunology Sciences, with minor modifications for detecting Tfh cells.Details of the gating strategy are in Supplementary Figure 2.

Table 4 . Associations of the immune cell types with the patient clusters.
(Supplementary Table4is provided as a separate Microsoft Excel sheet) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Table 5 . ISN/RPS classification of the SLE patient clusters.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) on this supplemental material which has been supplied by the author(s) on this supplemental material which has been supplied by the author(s) on this supplemental material which has been supplied by the author(s) placed doi: 10.1136/ard-2023-224537 -252.placeddoi:10.1136/ard-2023-224537 -252.placeddoi: 10.1136/ard-2023-224537 -252.:242 83 2024; Ann Rheum Dis , et al.Tanaka H